Effect Of ELTD1on Cardiac Hypertrophy In Response To Pressure Overload

Background—cardiac hypertrophy is a common process in various cardiac diseases, and is a major risk factor for heart failure. Epidermal growth factor (EGF), latrophilin and seven transmembrane domain-containing protein1(ELTD1) is developmentally upregulated in the heart. Furthermore, the extracellular domain of rat ELTD1possesses several common protein kinase phosphorylation sites, and the short cytoplasmic tail carries a tyrosine kinase phosphorylation site, which could be involved in cross-talk between signal transductions modules employing tyrosine kinases.Little is known about the relationship between ELTD1and cardiac diseases. Therefore, we aimed to clarify the role of ELTD1in pressure overload-induced cardiac hypertrophy.Methods1. ELTD1protein levels decrease in the ventricles of patients with end stage heart failure C57BL/6J (WT) mice male mice, aged between8and10weeks, were subjected to pressure overload by thoracic aortic banding. Ventricles of these mice were collected7,14,28, and56days post AB or immediately after sham surgery (n=6/group), and relative levels of ELTD1was examined by real-time PCR. The protein level of ELTD1of myocardial samples taken from failing human hearts (NYHA Ⅳ) and healthy controls (n=4/group) were assessed by western blotting.2. Effect of ELTD1deficiency on cardiac hypertrophyC57BL/6J (WT) and ELTD1knockout (KO)(C57BL/6J background) male mice, aged between8-10weeks, were subjected to pressure overload by thoracic aortic banding. Echocardiography and PV loop measurements were performed before mice were euthanized28days after the operations. Next, the hearts were harvested and randomly assigned for bio-molecular and histological analyses. Heart weight (HW), lung weight (LW), body weight (BW), and tibia length (TL) at28days after surgery. A single myocytes was measured using images captured from hematoxylin and eosin (H&E)-stained sections. Expression of the hypertrophy genes, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), α-myosin heavy chain (α-MHC),β-MHC, actin α1skeletal muscle (ACTA1) and sarco/endoplasmic reticulum Ca2+-transport ATPase2α (SERCA2α) were measured by RT-PCR.The cardiac collagen volume was calculated as the ratio of the sum of the total collagen area to the sum of the total collagen and no collagen areas in the entire visual field of the section as determined by picrosirius red (PSR) staining. Pro-collagen type Ⅰα1(Collal), pro-collagen type Ⅲ α1(Co13α1), transforming growth factor-β1(TGFβ1), connective tissue growth factor (CTGF), matrix metalloproteinase2(MMP2), and MMP9were measured by RT-PCR to elucidate the underlying mechanism for fibrosis.3. Effect of ELTD1on MAPK and Akt activationTotal protein samples were extracted from left ventricles. Western blots were used to detect total and phosphorylated levels of proteins in MAPKs and PI3K-Akt-mTOR/GSK3signaling in four groups (n=6/group).The GAPDH protein was used as the endogenous control.Results1. ELTD1was high expressed in hearts of mice and its expression was significantly changed after pressure overloadBy assessing the expression pattern in various tissues, we found a high protein level of ELTD1in the heart of C57BL/6J mice.The levels of ELTD1mRNA reached a maximum at7days after AB and then decreased. Ventricular tissue from failing hearts exhibited a2-to3-fold decrease in the ELTD1protein level relative to those from normal donors.2. ELTD1deficiency exacerbates cardiac hypertrophy after pressure overloadAB induced a significant increase in HW/BW and HW/TL in both WT and KO mice, and the increase was more pronounced in KO mice than in WT mice. Larger CSA accompanied by thicker wall thickness and more serious dilated chamber were shown in KO than those of WT after AB. AB induced a significant increase in HW/BW and HW/TL in both WT and KO mice, and the increase was more pronounced in KO mice than in WT mice, paralleled by a strong reduction in expression of the hypertrophy genes β-MHC and SERCA2α,and an increase in the expression of ANP, BNP, α-MHC, β-MHC, ACTA1and SERCA2α. AB significantly impaired cardiac function as evidenced by the markedly reduced dP/dtmax and dP/dtmin as compared to sham-operation. ELTD1deficiency aggravated the depression in the systolic function (dP/dtmax, EF, FS, cardiac output and stroke volume) after pressure overload, and similar results were observed for the diastolic function. PSR staining revealed striking perivascular and interstitial fibrosis in ventricles in the KO-AB mice compared to WT-AB. The mRNA levels of pro-collagen type Ⅰ al (Collα1), pro-collagen type Ⅲ α1(Col3α1), and fibronectin were significantly higher in KO mice than in WT mice after AB. TGFβ1, TGFβ2, and CTGF demonstrated exaggerated responses in KO mice relative to WT after AB. AB significantly increased the expression of MMP9and MMP2, but no difference was found in KO and WT after AB or sham operation3. Effect of ELTD1on MAPKs andPI3K-Akt activationAB promoted the phosphorylation of MEK1/2-ERK1/2, JNK1/2, and p38-MAPK in KO and WT mice. Deletion of ELTD1further increased activation of MRK1/2-ERK1/2and JNK1/2after AB. Notably, the phosphorylation of p38-MAPK was not affected by the absence of ELTD1. Our data showed that the activation of Akt was amplified by AB, but no difference was found between the two groups after AB.Conclusions—ELTD1deficiency exacerbates cardiac hypertrophy and cardiac function induced by AB-induced pressure overload by promoting both cardiomyocyte hypertrophy and cardiac fibrosis. These effects have been suggested to originate from the activation of the ERK and JNK pathways, suggesting that ELTD1is a potential target for therapies that prevent the development of cardiac disease.